The rearrangement of T cell receptor (TCR) gene segments occurs in a regulated fashion during T lymphocyte development. The objective of this proposal is to establish the molecular basis for developmentally regulated TCR gene rearrangement, with a specific focus on the TCR alpha/delta locus. A transgenic mouse model will be used to study V-D-J rearrangement of a human TCR delta gene minilocus. This minilocus is composed of two discrete regulatory domains. The TCR delta enhancer (E/delta) controls one of these domains, thereby regulating the V-D to J step of transgene rearrangement. Minilocus rearrangement will be compared under the control of E/delta and E/alpha to ask whether regulated activation of transcriptional enhancers is responsible for establishing the developmental timing and lineage specificity of gene rearrangement at the TCR alpha/delta locus. A role for lineage specific silencers of E/alpha will also be tested. A cis-acting element of E/delta called deltaE3 is essential for transcriptional activation, and two transacting factors, CBF and c-Myb, regulate enhancer activity through this element. Minilocus rearrangement will be assayed under the control of E/delta mutants with disrupted binding sites for CBF and c-Myb to assess the roles of these factors in developmental regulation of rearrangement. Minilocus accessibility, methylation and transcription will be evaluated to explore the basis for enhancer control of rearrangement, and to obtain additional physical and functional data that tests a two domain model of minilocus control. A novel insulator is predicted to delimit the boundary between two regulatory domains of the minilocus. The insulator will be identified by testing its ability to transcriptionally insulate promoters from enhancers on chromosomally integrated substrates. A novel role for insulators in enhancer-dependent developmental targeting of V-D-J rearrangement will be assessed by the analysis of transgenic mice. Differential expression of c-Myb in thymocytes and peripheral T cells implies a unique role for c-Myb in regulating E/delta function in developing thymocytes, and suggests distinct regulation of E/delta activity through deltaE3 and other enhancer elements in immature and mature T cells. Developmental control of E/delta and deltaE3 activity will be measured using transgenic reporter constructs, and developmental control of E/delta and deltaE3 occupancy will be measured using minilocus transgenic mice.